Validation of a set of reference genes to study response to herbicide stress in grasses

Abstract

BackgroundNon-target-site based resistance to herbicides is a major threat to the chemical control of agronomically noxious weeds. This adaptive trait is endowed by differences in the expression of a number of genes in plants that are resistant or sensitive to herbicides. Quantification of the expression of such genes requires normalising qPCR data using reference genes with stable expression in the system studied as internal standards. The aim of this study was to validate reference genes in Alopecurus myosuroides, a grass (Poaceae) weed of economic and agronomic importance with no genomic resources.ResultsThe stability of 11 candidate reference genes was assessed in plants resistant or sensitive to herbicides subjected or not to herbicide stress using the complementary statistical methods implemented by NormFinder, BestKeeper and geNorm. Ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase were identified as the best reference genes. The reference gene set accuracy was confirmed by analysing the expression of the gene encoding acetyl-coenzyme A carboxylase, a major herbicide target enzyme, and of an herbicide-induced gene encoding a glutathione-S-transferase.ConclusionsThis is the first study describing a set of reference genes (ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase) with a stable expression under herbicide stress in grasses. These genes are also candidate reference genes of choice for studies seeking to identify stress-responsive genes in grasses.

Selection and evaluation of potential reference genes for gene expression analysis in Avena fatua

“…Based on the rankings from RefFinder, which integrates outcomes of the four major statistic algorithms (ΔCt methods, geNorm, Normfinder, and Bestkeeper), and it also assigns an appropriate weight to an individual gene and calculates the geometric mean of their weight, GAPDH and EF1α had a good performance under specific conditions. Of these reference genes tested (GAPDH, EF1α, 18S, 28S, and ACT) in A. fatua varied greatly, GAPDH and EF1α were recommended as the most suitable reference genes while 18S was ranked as the less suitable reference genes under the majority of the experimental conditions in our results, which was consistent with the other studies examining reference gene expression (Petit et al 2012;Duhoux & Délye 2013). However, some reports indicated 18S was recommended to validate gene expression data in Solanum melongena L. or Oryza sativa L. (Kim et al 2003;Gantasala et al 2013).…”

Differential Expression of Genes Associated With Degradation Enhancement of Imazethapyr in Barnyardgrass (Echinochloa crus-galli)

“…A number of studies demonstrate that the expression of reference genes widely used in this type of study can vary considerably with experimental conditions, tissues and species (Thellin et al, 1999;Stürzenbaum & Kille, 2001). The analysis of the stability in the expression of reference genes in weeds responding to herbicide stress has already been performed in A. myosuroides for the acetyl CoA carboxylase (ACCase) inhibitors herbicides (Petit, Permin, Heydel, & Délye, 2012), and in L. rigidum for ALS inhibitors herbicides (Duhoux & Délye, 2013). In these studies, the most stable reference gene for A. myosuroides and L. rigidum were genes coding for tubulin and CAP proteins, respectively.…”

Real Time PCR: the Use of Reference Genes and Essential Rules Required to Obtain Normalisation Data Reliable to Quantitative Gene Expression

“…The BestKeeper use raw Ct data and determines the most stably expressed genes based on a correlation coefficient (r) of the BestKeeper Index (BI) and standard deviation, whereas BI is the geometric mean of Ct values of best reference genes. Hence, this program relies on the "r" and "SD" values, and the higher the "r" value, the most stable is the gene; otherwise, the lower the standard deviation value, the most stable is the gene (Pfaffl et al, 2004;Demidenko et al, 2011;Niu et al, 2011;Petit et al, 2012).…”